CN109374900B

CN109374900B - Vaccine antibody detection method based on detection card

Info

Publication number: CN109374900B
Application number: CN201811116318.0A
Authority: CN
Inventors: 邱灵冰
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-09-25
Filing date: 2018-09-25
Publication date: 2022-05-31
Anticipated expiration: 2038-09-25
Also published as: CN109374900A

Abstract

The invention discloses a vaccine antibody detection method based on a detection card. The detection card comprises a test strip, and the test strip is provided with a sample cushion layer, a colloidal gold layer, a nitrocellulose membrane and a water absorption layer. The invention can specifically detect the specific antibodies generated by different vaccines by using a colloidal gold immunochromatographic technique. The vaccine antibody detection card is convenient, the sample dosage is less, the vaccine antibody detection can be realized only by 50 mu L of serum, plasma or whole blood, the operation is simple, the detection speed is high, the field is not limited, and the vaccine antibody detection card is suitable for clinical and community popularization.

Description

Vaccine antibody detection method based on detection card

Technical Field

The invention belongs to the technical field of immunological detection, and particularly relates to a vaccine antibody rapid and efficient method based on a detection card.

Background

Vaccines are preventive biological products used for human vaccination in order to prevent and control the occurrence and prevalence of infectious diseases. Biologics for vaccination include vaccines, bacterins and toxoids. Wherein the vaccine is prepared from germs; the vaccine is prepared from virus, rickettsia and spirochete and is called vaccine collectively. The vaccine is an automatic immune preparation for preventing infectious diseases, which is prepared by artificially attenuating and inactivating pathogenic microorganisms (such as bacteria, rickettsia, viruses and the like) and metabolites thereof or by utilizing genetic engineering and other methods. After the pathogenic bacteria without harm are inoculated, the immune system can generate certain protective substances, such as immune hormone, active physiological substances, special antibodies and the like; when the body is again exposed to such pathogenic bacteria, the immune system of the body is rapidly activated, producing specific antibodies to protect the body.

Because of individual constitution difference or whether the vaccine itself and other objective factors, whether the effective antibody is generated in the organism after the vaccine injection, no suitable convenient, simple and quick detection method exists at present. The antibody generated by immune system of vaccine can be detected by colloidal gold immunochromatography.

The colloidal gold immunochromatographic assay is a novel immunolabeling technology which uses colloidal gold as a tracer marker to apply to antigen and antibody. The advantages are that:

1. the use is convenient and quick, the method is not limited to places, and all reactions can be completed within 15 minutes;

2. the cost is low, and special instruments and equipment are not needed;

3. the application range is wide, and the method can adapt to various detection conditions;

4. multiple tests can be carried out, if the samples are difficult to obtain, the multiple tests can save the samples and reduce the cost;

5. the marker is stable, and the marked sample is stored for more than two years at 4 ℃ without signal attenuation;

6. the colloidal gold is red, and a color development reagent is not required to be added, so that the steps of an enzyme-labeled carcinogenic substrate and a stopping solution are omitted, and the colloidal gold is harmless to a human body.

At present, the prior art has no method for detecting the antibody conveniently, simply, quickly and with high sensitivity.

Disclosure of Invention

The invention aims to provide a vaccine antibody detection method based on a detection card, which comprises the following steps:

(1) filtering to remove impurities in the sample to be detected to obtain a sample feeding liquid,

(2) reacting the sample solution with colloidal gold particles under conditions suitable for the reaction to carry out the complexing, wherein the average diameter of the colloidal gold particles is 15-50 nm;

(3) filtering to remove at least part of the colloidal gold particles which are not combined with the antibody in the step (2) to obtain a chromatographic solution;

(4) carrying out chromatography on the chromatography liquid in the step (3), and enabling the antibody to be bound to the detection line T line;

(5) judging whether the result in the step (4) is credible through the C line as a comparison.

Preferably, in the vaccine antibody detection method of the present invention, the detection card comprises a test strip, and the test strip is provided with a sample pad layer and a first filter layer having a pore size that allows antibody molecules to pass through but does not allow molecular aggregates to pass through, and step (1) is performed through the first filter layer.

Preferably, in the vaccine antibody detection method of the present invention, the test strip further includes a colloidal gold layer, and the colloidal gold layer includes a glass fiber layer and a colloidal gold marker dispersed in the glass fiber layer and binding to the vaccine specific antibody, the colloidal gold marker is a conjugate of an anti-antibody against the vaccine antibody to be detected and colloidal gold particles, and step (2) is performed on the colloidal gold layer.

Preferably, in the vaccine antibody detection method of the present invention, the test strip further includes a second filter layer having a pore size that allows small molecules to pass through but does not allow antibody molecules to pass through, at least a portion of the second filter layer is disposed below the colloidal gold layer, and step (3) is performed through the second filter layer.

Preferably, in the vaccine antibody detection method of the present invention, the test strip further comprises a nano nitrocellulose membrane, on which a T line and a C line are disposed, wherein the T line is coated with an antigen of the target vaccine, and the C line is coated with a goat anti-mouse IgG antibody, and step (4) and step (5) are performed on the nano nitrocellulose membrane.

Preferably, in the vaccine antibody detection method of the present invention, the test strip further includes a support layer and a water-absorbing layer, and the sample pad layer, the colloidal gold layer, the nitrocellulose membrane, and the water-absorbing layer are sequentially disposed on the same side of the support layer along the length direction thereof in a manner of being connected to each other.

Preferably, in the vaccine antibody detection method of the present invention, the sample pad layer, the colloidal gold layer, and the nitrocellulose membrane are adjacently connected in sequence, and there is no overlap between the sample pad layer and the nitrocellulose membrane and between the colloidal gold layer and the nitrocellulose membrane, and the first filter layer is disposed above the sample pad layer, and the second filter layer is disposed below the colloidal gold layer.

Preferably, in the vaccine antibody detection method of the present invention, the average pore size of the first filtration layer is 250-500nm, and the average pore size of the second filtration layer 2221 is 50-70 nm.

Preferably, in the vaccine antibody detection method of the present invention, the second filter layer is made of a water-absorbent material.

Preferably, in the vaccine antibody detection method of the present invention, an interface between the colloidal gold layer and the second filter layer is higher than an interface between the support layer and the nitrocellulose membrane.

The beneficial technical effects are as follows:

the invention can specifically detect the specific antibodies generated by different vaccines by using a colloidal gold immunochromatographic technique. The vaccine antibody detection card is convenient, the sample dosage is less, the vaccine antibody detection can be realized only by 50 mu L of serum, plasma or whole blood, the operation is simple, the detection speed is high, the field is not limited, and the vaccine antibody detection card is suitable for clinical and community popularization.

Preferably, the invention filters impurities such as molecular groups or large particles through the first filter layer, so that the impurities are prevented from entering the nitrocellulose membrane, and the influence of the substances on detection is avoided. In addition, the second filter layer filters out the colloidal gold particles which are not combined with the antibody and other impurities such as small molecules, and the impurities are prevented from entering the nitrocellulose membrane. In the conventional method, these impurities can fill the pores of the nitrocellulose membrane, thereby being disadvantageous to the movement of the antigen-antibody conjugate to be detected, and the colloidal gold particles to which the antibody is not bound affect the reaction strength at the position of the detection line.

Preferably, the nano nitrocellulose membrane is adopted to replace the conventional general nitrocellulose membrane, and by adopting the technical scheme of the invention, the nano nitrocellulose membrane does not reduce the detection sensitivity and still has excellent effect. This is in contrast to what is generally understood in the art.

Drawings

FIG. 1 is a perspective view of an exemplary vaccine antibody detection card of the present invention.

FIG. 2 is a diagram of a reagent strip of an exemplary vaccine antibody test card of the present invention.

FIG. 3 is a partial block diagram of a reagent strip of another exemplary vaccine antibody test card of the present invention.

Description of reference numerals:

the vaccine antibody detection card 100, the card cover 110, the card body 120, the reagent strip 130, the sample application hole 111, the observation window 112, the

sample pad layers

121 and 221, the

colloidal gold layers

122 and 222, the

nitrocellulose membranes

123 and 223, the water absorption layer 124, the supporting

layers

125 and 225, the first filter layer 2211 and the second filter layer 2221.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

Preparation example

This embodiment is a method for preparing an exemplary test card for use in the present invention, which comprises the following steps:

(1) preparation of colloidal gold

According to different types of vaccines needing to be detected, colloidal gold particles with proper sizes can be selected, and the preparation of the colloidal gold particles with the sizes of 15nm, 18nm-20nm, 30nm or 50nm is as follows: 100ml of 0.01% HAuCl4 aqueous solution was taken and boiled. Adding 1% trisodium citrate water solution 4ml, 2.5ml, 1ml or 0.75ml as required, and boiling for about 5min to obtain orange red color. The colloidal gold particles thus prepared were 15nm, 18-20nm, 30nm and 50nm, respectively. Cooling for later use;

(2) labeling of colloidal gold

Colloidal gold-labeled mouse anti-human immunoglobulin antibody: taking 50mL of the colloidal gold solution prepared in the step (1), and adding 0.1mol/L K₂CO₃Adjusting pH to 8.9-9.2, and adding 4-6mL mouse-human immunoglobulin with concentration of 0.8-1.2mg/mLStirring the antibody for 2-5 minutes, adding 4-6mL of PEG20000 aqueous solution with the mass fraction of 0.8-1.2%, centrifuging at 15000r/min for 35-45 minutes, sucking the supernatant, suspending the precipitate in 0.4-0.6mg/mL of PEG20000 aqueous solution, preserving with 0.5mg/mL of sodium azide, and storing at 3-5 ℃;

(3) solid phase of colloidal gold

Equivalently mixing the colloidal gold marker solutions prepared in the steps (1) and (2), and carrying out solid phase on the colloidal gold layer to obtain a glass fiber gold marker pad;

(4) coated nitrocellulose membrane

4.1C-coated antigen: diluting different vaccine antigens to 0.8-1.2mg/mL by using PBS buffer solution with the pH value of 7.2-7.5, and spraying the different vaccine antigens on a nitrocellulose membrane by using a membrane spraying machine at the pump speed of 16mm/s and the membrane speed of 320mm/s to form a target vaccine antigen detection line;

4.2T-coated goat anti-mouse IgG antibody: diluting the goat anti-mouse IgG antibody to 1.4-1.6mg/mL by using PBS buffer solution with pH of 7.2-7.5, and spraying the diluted goat anti-mouse IgG antibody on the cellulose nitrate membrane prepared in the step 4.1 by using a membrane spraying machine at a pump speed of 16mm/s and a membrane speed of 320mm/s to form a goat anti-mouse IgG antibody quality control line;

4.3 drying the nitrocellulose membrane prepared in the step 4.2 in an oven at 37 ℃ for 1.5-2 hours;

(5) preparation of test paper strip

5.1 sticking the nitrocellulose membrane prepared in the step 4.3 at the middle position of the PVC plate;

5.2 pasting absorbent paper above the position of the fixed nitrocellulose membrane of the PVC plate, wherein the absorbent paper covers 1.0 mm of the edge of the nitrocellulose membrane;

5.3 adhering the glass fiber gold label pad and the sample pad of the colloidal gold marker prepared in the step (3) below the position of the nitrocellulose membrane fixed by the PVC plate, wherein the glass fiber gold label pad covers the nitrocellulose membrane by 1.0 mm,

5.4 flattening the product obtained in the step 5.3, and cutting the product into test strips with the width of 5.0 mm;

(7) and taking the card cover and the card body, placing the prepared test strip in the groove of the card body, and buckling the card cover and the card body to obtain the vaccine antibody detection card.

Example 1

This embodiment is an exemplary test card 100 for use with the present invention. As shown in fig. 1 and 2, the vaccine antibody detection card 100 includes a card cover 110, a card body 120, and a reagent strip 130.

The card cover 10 is provided with a sample application hole 111, an observation window 112 and a sample number mark (not shown), and the card body 120 is provided with a groove for placing the reagent strip 130. The card cover 110 and the card body 120 form a closed sealed box by snap-fitting.

The test strip 130 is provided with a sample pad layer 121, a colloidal gold layer 122, a nitrocellulose membrane 123 and a water absorbing layer 124, wherein the sample pad layer 121, the colloidal gold layer 122, the nitrocellulose membrane 123 and the water absorbing layer 124 are respectively arranged on the same side of a PVC plate as a supporting layer 125, and the sample pad layer 121, the colloidal gold layer 122, the nitrocellulose membrane 123 and the water absorbing layer 124 are sequentially arranged in an interconnected manner along the length direction of the supporting body 125. A water absorbing layer 124 is stuck on one end of the nitrocellulose membrane 123, and the water absorbing layer 124 covers 1.0 mm above the edge of the nitrocellulose membrane 123, namely the water absorbing layer 124 and the nitrocellulose membrane 123 are overlapped by 1.0 mm. A colloidal gold layer 122 is pasted above the other end of the nitrocellulose membrane 123, and the colloidal gold layer 122 covers 1.0 mm above the edge of the nitrocellulose membrane 123, i.e. the weight of the colloidal gold layer 122 and the nitrocellulose membrane 123 is 1.0 mm. The sample pad layer overlaps the other end of the colloidal gold layer by about 1.0 mm. The colloidal gold layer contains colloidal gold particles having a diameter of 50 nm.

The colloidal gold layer 122 is a glass fiber layer that contains a colloidal gold label that binds a vaccine-specific antibody. In this embodiment, the colloidal gold label is a conjugate of an anti-antibody of the vaccine-specific antibody and the colloidal gold label. The nitrocellulose membrane 123 is provided with an antigenicity detection line C coated with a target vaccine and a quality control line T coated with a goat anti-mouse IgG antibody.

The sample application hole 111 of the vaccine antibody detection card 100 in this embodiment is provided at a position corresponding to the sample pad 121, and the observation window 112 is provided at a position corresponding to the nitrocellulose membrane 123.

In this embodiment, on the basis of fig. 1 and 2, a first filter layer 2211 and a second filter layer 2221 are further included. Fig. 3A shows that the second filter layer 2221 has the same size as the colloidal gold layer 222, and the support 225 has a groove having the same size as the second filter layer 2221 for fixing or mounting the second filter layer 2221. Fig. 3B shows that the second filter layer 2221 has the same size as the sum of both the sample pad layer 221 and the colloidal gold layer 222, and the second filter layer 2221 is located below the sample pad layer 221 and the colloidal gold layer 222. Fig. 3C shows the second filter layer 2221 disposed over the support layer 225, and the size of the second filter layer 2221 is the same as the support layer 225.

In this embodiment, the first filter layer has an average pore size of 250-500nm and the second filter layer 2221 has an average pore size of 50-70 nm. Preferably, the second filter layer 2221 is made of a material having strong water absorption. More preferably, the interface between the colloidal gold layer 222 and the second filter layer 2221 layer is higher than the interface between the support 225 and the nitrocellulose membrane.

Because the second filter layer 2221 can filter and remove most of the colloidal gold particles not bound with the antibody, the movement of antigen-antibody binding molecules in the nitrocellulose membrane is facilitated, and the removed colloid not bound with the antibody does not appear in the detection line, so that the detection accuracy is greatly improved.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

Claims

1. A vaccine antibody detection method based on a detection card is characterized by comprising the following steps:

(1) filtering and removing impurities in a sample to be detected through a first filter layer to obtain a sample inlet liquid, wherein the first filter layer is provided with a pore diameter which allows antibody molecules to pass through but cannot allow molecular aggregates to pass through;

(3) filtering and removing at least part of the colloidal gold particles which are not bonded with the antibody in the step (2) through a second filtering layer to obtain a chromatographic solution, wherein the second filtering layer has a pore size which allows small molecules to pass through but cannot allow antibody molecules to pass through;

(5) judging whether the result in the step (4) is credible through the line C as a comparison;

the detection card comprises a test strip, the test strip is provided with a sample cushion layer and a colloidal gold layer, at least one part of the second filter layer is arranged below the colloidal gold layer, the first filter layer is arranged above the sample cushion layer, and the second filter layer is arranged below the colloidal gold layer.

2. The method for detecting the vaccine antibody according to claim 1, wherein the colloidal gold layer comprises a glass fiber layer and a colloidal gold marker dispersed in the glass fiber layer and binding to the vaccine specific antibody, the colloidal gold marker is a combination of an anti-antibody against the vaccine antibody to be detected and colloidal gold particles, and step (2) is performed on the colloidal gold layer.

3. The vaccine antibody detection method according to claim 1, wherein the test strip further comprises a nano-nitrocellulose membrane on which a T-line and a C-line are disposed, wherein the T-line is coated with an antigen of a vaccine of interest, and the C-line is coated with a goat anti-mouse IgG antibody, and the steps (4) and (5) are performed on the nano-nitrocellulose membrane.

4. The vaccine antibody detection method according to claim 3, wherein the test strip further comprises a support layer and a water-absorbing layer, and the sample pad layer, the colloidal gold layer, the nitrocellulose membrane and the water-absorbing layer are sequentially arranged on the same side of the support layer along the length direction thereof in an interconnected manner.

5. The method for detecting the vaccine antibody according to claim 4, wherein the sample pad layer, the colloidal gold layer and the nitrocellulose membrane are adjacently connected in sequence, and there is no overlap between the sample pad layer and the nitrocellulose membrane and between the colloidal gold layer and the nitrocellulose membrane.

6. The method for detecting vaccine antibodies as claimed in claim 5, wherein the first filter layer has an average pore size of 250-500nm and the second filter layer 2221 has an average pore size of 50-70 nm.

7. The method for detecting vaccine antibodies according to claim 6, wherein the second filter layer is made of a water-absorbent material.

8. The vaccine antibody detection method according to claim 7, wherein an interface between the colloidal gold layer and the second filter layer is higher than an interface between the support layer and the nitrocellulose membrane.